Objective To sequence and analyze the full-length genome of one HEV strain,W2-1 isolated from a sporadic hepatitis E patient hospitalized in 1999 in Xinjiang,China.Methods Nested RT-PCR assays with 4 sets primers were used to amplify the entire genome.The PCR products were purified and sequenced.The full-length genome was acquired by assembling the fragmental sequences using the DNAstar 5.01 software.The genome of W2-1 was analyzed by comparing with the reference HEVs from GenBank.Results The complete genome of W2-1 is 7212 nt in length,including three open reading frames (ORF1-3) with 5079,1980 and 345 nt respectively,27 nt 5'UTR and 83 nt 3'UTR,and a 3' poly A tail.Phylogenetic analysis based on full-length genome showed that W2-1 belonged to genotype 1,subtype 1b.W2-1 had high homology with the HEV strains isolated in the large hepatitis E epidemic in Xinjiang in 1987-1989,sharing 97.2％-98.5％ nucleotide identity in the full length genome.W2-1 also showed high homology with 1b strains isolated in China after 2000,with 97.6％-99.2％ nucleotide identity.The specific amino acid sites in ORF1-3 proteins that distinct between genotype 1 HEV and the potential zoonotic strains did not change in W2-1.Conclusion W2-1 belongs to subtype 1b.The study indicates subtype 1b HEV has been circulating in China in a long period after hepatitis E outbreak in Xinjiang in 1986-1989.The amino acids of ORF1-3 of subtype 1b are conserved.

Objective To investigate the effects of adenosine (Ado) on myocardiac electrophysiology in simulated ischemia and reperfusion in guinea-pig ventricular myocytes. Methods Electrical activity was recorded using standard intracellular microelectrode technique. Right ventricle was superfused with simulated ischemic Tyrode's solution for 15 min, and reperfued with normal Tyrode's solution for 30 min. Results The results showed Ado had no measurable effects on guinea-pig ventricular myocytes in normal Tyrode's solution. In the presence of Ado, maximal diastolic potential tended to be more depolarized during ischemia, and action potential (AP) parameters were abbreviated greatly in a concentration-dependent manner. Especially, the concentration of Ado 100 μmol·L-1 had significant effects on AP parameters in ischemic phase [APD30, APD50, and APD90 reduced by (86 ± 8) ％ versus (65± 6) ％, (70± 7)％versus (50±6)％, and (60±6)％versus (42±4)％ for control after 15 min, P＜0.05]. During reperfusion, AP parameters did not completely return to initial values in presence of Ado. This study illustrated that Ado significantly decreased incidence of arrhythmias induced by ischemia and reperfusion (in presence of Ado 100 μmol·L-1, the incidence of DAD decreased by 17％ versus 82％ for control during reperfusion). Conclusion Ado has no significant effects on guinea-pig ventricle in normal conditions, abbreviates greatly AP parameters during ischemia with a concentration-dependent manner, and has marked antiarrhythmic effects in ischemia and reperfusion.

Objective To analyze the practicability of using ELISA kit for the detection of hepati-tis E virus antigen ( HEV-Ag) in plasma donations and Biomex HEV seroconversion panels. Methods The HEV-Ag positive samples were screened out from 36 340 donated blood plasma samples. Real-time fluores-cent PCR was performed for the detection of HEV RNA in HEV-Ag positive samples. The open reading frame 2 (ORF2) in HEV RNA was amplified by nested RT-PCR and the amplified products were confirmed by sequencing analysis. Phylogenetic tree was constructed for HEV genotyping. Five Biomex HEV serocon-version panels were used in this study for the detection of HEV-Ag, anti-HEV antibody and HEV RNA as well as the correlation analysis between HEV-Ag and HEV RNA. Results Twenty-six out of 36 340 plasma samples (0. 07%) were positive for HEV-Ag. Of the 26 samples, 25 samples were positive for HEV RNA as indicated by the results of nested RT-PCR and 23 positive samples were confirmed by sequencing analysis. The positive rate of HEV RNA in blood plasma donators was 1 ∶ 1 580 (0. 06%). There were 17 samples of genotype 1 (74%) and 6 samples of genotype 4 (26%) according to the phylogenetic tree analysis. All of the HEV-Ag positive samples were also positive for HEV RNA as indicated by the analysis of Biomex sero-conversion panels. HEV-Ag was consistent with the peak of the HEV RNA concentration. Conclusion A close relationship between HEV-Ag and HEV RNA was observed. HEV-Ag screening could be used as a measure to reduce the risk of HEV transmission by blood transfusion.

Objective To study the in vivo expression and biodistribution of Ad5-Fluc (Adenovirus carrying firefly luciferase genes) in mice.Methods The recombinant Ad5-Fluc virus was constructed and infected to BALB/c or nude mice through three different routes.The protein expression level,tissue distribution and the characteristics of infection were analyzed by in vivo bioluminescence imaging technology.Results Compared to other two routes,the BALB/c mice infected through muscular route had the longest expression cycle (over 60 days) and the highest expression level,while the virus was transferred into the liver and spleen after infection.The nude mice had a significantly extended expression cycle than BALB/c mice.Moreover,the characteristic of liver tropism was eliminated after Ad5 F35 infection in mice,while maintained similar expression efficiency.Conclusion Due to the highest expression efficiency,the muscular route would be the optimal route for Ad5 vector based vaccination.In addition,Ad5F35 virus could become an ideal alternative vaccine vector for eliminating the liver tropism.

Objective To compare cellular immune responses in mice elicited by Chinese different AIDS candidate vaccines.Methods According to their different immunization procedures,BALB/c mice were immunized with 6 AIDS candidate vaccines,separately.Spleen cells were isolated for the detection of cellular immune response to HIV-specific peptides using enzyme-linked immunosorbent spot(ELISPOT)assay and intracellular cytokine staining(ICS)method.Results AIDS vaccines were evaluated by using potential T-cell epitopes(PTE)Gag,Env and Pol peptides pool and ELISPOT.The positive conversion rates for cellular immune response of 1#-6# vaccines fluctuated from 70% to 100%.The vaccine-induced cellular immune responses to specific peptides pool are different not only in magnitude but also in breadth.The Th1type cytokines,IFN-γand IL-2,were detected with ELISPOT in 1# and 2# vaccines.The productions of IFN-γand IL-2 induced by both of the two vaccines showed a moderate correlation(r1 =0.62,P1 ＜0.01 ;r2=0.79,P2 ＜ 0.01).The positive conversion rate of IFN-γ secreting cells of 1 # vaccine was 66.7%(10/15)mice detected with both ELISPOT and ICS.And the results tested by ELISPOT and ICS showed moderate correlation(r = 0.55,P ＜ 0.05).Conclusion The magnitude and breadth of cellular immune responses induced by different AIDS candidate vaccines are different.Being induced by different AIDS candidate vaccines,the IFN-γand other Th1 type cytokines detected by ELISPOT or ICS could be used to evaluate the cellular immune responses in mice.

Objective To establish a high throughput phenotypic test for the detection of drug re-sistance in human immunodeficiency virus(HIV)strains. Methods The gene encoding luciferase was in-activated through restriction enzyme digestion and ligation. LacZ gene was used to replace the genes encoding original protease and reverse transcriptase. pol genes were amplified from pSG3△env plasmid and cloned in-to a new backbone plasmid through infusion. The factors that might affect the results of the test were opti-mized. Results The parental backbone plasmid pNL4-3. Lac was constructed,of which the gene encoding luciferase was inactivated and bearing the LacZ gene instead of genes encoding protease and reverse tran-scriptase. Several influential factors including cell numbers(10 000 / well),virus inoculation(200 TCID50 /well)and the concentration of DEAE-dextran(15 μg/ ml)were optimized. The reproducibility of this test was confirmed by testing 12 anti-HIV drugs against 2 pseudovirus strains 8 times,presenting the coefficient of variations(CVs)from 4. 32% to 28. 46% . Six types of pseudovirus were constructed and tested against the 12 anti-HIV drugs,the results of which were compared with those by using the pSG3△env-based pseud-ovirus test. The results of the two tests presented good consistency. Conclusion The high throughput phe-notypic test based on pNL4-3. Lac plasmid,combining the advantages of pSG3△env and pNL4-3 systems, could be used to analyze the drug resistance patterns of HIV-1 infectors and screen new drugs for antiretrovi-ral therapy in a rapid and effective way.

The Long-term Thromboembolic Event Analysis in Atrial Fibrillation Patients With Radiofrequency Catheter Ablation
Objective: To observe the thromboembolic event in atrial fibrillation (AF) patients with long-term successful radiofrequency catheter ablation (RFCA), and to study the relationship between thromboembolic event and CHA2DS2-VASC score in order to guide the anticoagulation strategy for AF patients.
Methods: A total of 321 AF patients who received RFCA in our hospital from 2000-01 to 2009-05 were studied. There were 261 patients with paroxysmal AF and 60 with persistent AF, they were followed-up for (66.7±26.9) months. The patients were divided into 2 groups according to AF recurrence condition as Non-recurrence group, n=204 and Recurrence group, n=117. The relationship between thromboembolic event and CHA2DS2-VASC score was studied.
Results: The Non-recurrence group had significantly lower rate of thromboembolism than that in Recurrence group (1.96% vs 7.69%), P=0.017. In both groups, the patients with CHA2DS2-VASC score < 2 had much lower rate of thromboembolism than those with CHA2DS2-VASC score ≥ 2, (0% vs 5%), P=0.023 and (4.45% vs 17.24%), P=0.041. The patients with CHA2DS2-VASC score<2 in Non-recurrence group had lower rate of thromboembolism than those in Recurrence group (0%vs 4.45%), P=0.029. The rate of thromboembolism had no statistic meaning between 2 groups in patients with CHA2DS2-VASC score≥2 (5%vs 17.24%), P=0.054.
Conclusion: The AF patients who received RFCA without AF recurrence in long-term follow-up had the lower rate of thromboembolic event, CHA2DS2-VASC score was important for evaluating such event. The patients with CHA2DS2-VASC score < 2 could consider stopping warfarin anticoagulation, while the patients with CHA2DS2-VASC score ≥ 2 might be beneifted for warfarin anticoagulation.

Objective To study the effects of dichloroacetate (DCA) on cell colony-forming,cell invasion and cell migration of the bladder cancer cells and to study the underlying mechanism.Methods The bldder cancer cells T24 were randomly divided into two groups:the observation group and the control group.Cells in the observation groups were treated with 5 mmol/L,10 mmol/L and 20 mmol/L dichloroacetate,and the control group was treated with the same amount of dimethyl sulfoxide.Colony formation assays were detected with Giemsa staining.Cell wound scratch assay and Transwell assay were applied to evaluate the ability of the T24 cell invasion and migration.Realtime PCR and Western blot were applied to detect the expression of the epithelial-mesenchymal transition (EMT)-related marker,including E-cadherin,N-cadherin,vimentin,Snail and Slug.Results Compared with the control group,the colony formation assays of T24 cells constantly decreased along with the increased doses in the observation group(P ＜ 0.01).The cell wound scratch assay showed that the scratch width of the observation groups were significantly higher along with the increased doses and prolonged time than that in the control group (P ＜ 0.01).The transwell assay showed that the invasion ability of the observation groups were significantly discreased along with the increased doses than that in the the control group (P ＜ 0.01).The expression levels of E-cadherin mRNA and protein in combination the control group were higher than those in the the observation groups (P ＜ 0.05).However,the expression levels of N-cadherin,vimentin,Snail and Slug mRNAs and proteins in combination the control group were lower than those in the the observation groups (P ＜ 0.05).Conclusion Dichloroacetate can inhibit the colony-forming,invasion and migration of bladder cancer T24 cells,and its mechanism may inhibit the expression of epithelial mesenchymal transition in T24 cells by down-regulating the expression of nuclear transcription factor Snail and Slug.

Edong Healthcare Group is cited as an example to summarize its practice of centralized purchasing of supplies in the recent two years.In addition to a success in its process and mechanism, the authors analyzed some challenges for the purpose of furthering the group-based reform of public hospitals.

Objective: To investigate the presence of hepatitis E virus (HEV) RNA and HEV antigen (HEV-Ag) and to evaluate the infectivity of HEV in urine through a SPF rabbit model of HEV infection. Methods: Serum, fecal and urine samples collected from SPF rabbits with HEV infection were tested for viral and biochemical markers using real-time RT-PCR and ELISA. Liver and kidney biopsies were performed for observing histopathological changes and immunohistochemical staining. Rabbits were challenged with HEV isolated in urine samples to evaluate the infectivity. Results: Rabbit R1# that was injected with rabbit HEV presented viremia, fecal shedding of HEV, high serum level of HEV-Ag, elevated aspartate transaminase (AST) and alanine transaminase (ALT) and typical symptoms of hepatitis. Urine samples of rabbit R1# continued to be positive for HEV RNA and HEV-Ag. Ratios of HEV-Ag to RNA in urine samples of rabbit R1# were significantly higher than those in serum and feces samples. The parameters quantified in routine urinalysis remained within the normal ranges in rabbit R1#. However, pathological changes and the presence of HEV-Ag were observed in kidney tissues. Furthermore, serum and fecal samples that were collected from one of the two rabbits injected with rabbit R1# urine-derived HEV were HEV positive and the virus strains isolated form feces remained infective to rabbits. Conclusion HEV infection may result in kidney injury and the urine may pose a risk of transmission. HEV-Ag detection in urine may be valuable for the diagnosis of ongoing HEV infection.